Apples unpeeled: Proteome response to wounding

Infected apples

Apples are some of the easiest fruits to damage after they are picked. A small bruise can spread quickly into the fruit and nicks and cuts can become infected with moulds. Gray mould (Botrytis cinerea) and blue mould (Penicillium expansum) are particularly prevalent. It is very difficult to prevent damage like this to fruit during harvesting, packing and transport and it leads to large economic losses to food producers.

When fruit is wounded, it initiates inbuilt defence mechanisms. Various cellular processes kick in, supported by factors such as the production of lignin which help to reduce subsequent damage. Stress management is accompanied by changes in gene expression which, in turn, modify the collection of metabolites and proteins that are present. However, studies on the proteome response to wounding in apples are few and far between.

Now, European scientists have performed a study of the proteome changes in apples, specifically, the Golden Delicious variety, which occur after they have been wounded. Rosario Torres and colleagues from the IRTA research institute, Lleida, Lleida University, Spain and the Catholic University of Leuven, Belgium, argued that knowledge of the affected proteins will lead to better ways to reduce postharvest losses.

Getting to the core of the problem

A sterilised nail was used to nick 15 apples, after which they were inoculated with distilled water and stored for up to 2 days. They were tested after 24 and 48 hours, with those at time zero being used as the wounded control samples. Small cylinders of peel and fruit were cut out and the proteins were extracted for two-dimensional gel electrophoresis.

The protein spots were stained and those deemed to have changed in abundance between the controls and wounded apples were of key interest. However, proteins that were unaffected by wounding were also examined to see which functions they represented.

They were all identified by mass spectrometry, following in-gel digestion with trypsin and database searching. The resulting peptide masses were matched in several sequence databases, including one for the Malus variety and viridiplantae (green plants) to reveal the proteins present. Their functions were defined by gene ontology to help work out what happens after an apple is wounded.

Stress proteins

Only eight proteins were altered in abundance after damage and the team managed to identify five of these, which were localised around the wound. They were all involved in the stress response. In addition, 53 proteins that were unchanged after wounding were also identified from the average of 250 protein spots on each gel.

The abundance of thaumatin-like protein 1-a was increased after wounding, in line with reports that it was raised in the phloem exudates of wounded plants and might be involved in protecting peaches from damage during chilling. Large increases in Mal d 1-like protein and Mal d 1.03G of 2.70- and 5.98-fold, respectively, were also consistent with reports of this type of protein being induced by stress and disease.

Levels of the enzyme beta-glucanase fell after 24 hours. Enzymes of this type have been associated with the defence against fungal attack but they are also implicated in many other processes in fruit including ripening, seed germination and response to cold and ozone. In the case of the Golden Delicious apples, the researchers suggested that the decrease might be due to accelerated ripening of the fruit.

The fifth protein with altered abundance was flavoproteins WrbA-like protein, for which the researchers could offer no explanation at this stage.

Many of the proteins that were not altered after wounding were also associated with stress response, including others from the Mal d 1 group. So, it appears that some type of functional specialisation exists within this family for wound response. Heat shock proteins and other proteins normally associated with stress were also unchanged, but these are normally associated with cold shock.

This preliminary study on the response of apples to wounding has shed a little more light on their reaction to postharvest infection. Working with these altered proteins could lead to new ways to beef up their defence mechanisms.